Responses of Houttuynia cordata Thunb. to Tetracycline Stress

Yuanshuai WANG, Shiqiong LUO, Zhannan YANG*, Haitao YE, Qian DING

1. Key Laboratory for Information System of Mountainous Areas and Protection of Ecological Environment of Guizhou Province, Guizhou Normal University, Guiyang 550025, China; 2. School of Life Science, Guizhou Normal University, Guiyang 550025, China

Abstract [Objectives] To study responses of Houttuynia cordata Thunb. to tetracycline (TC) stress in a sterile environment. [Methods] Aseptic H. cordata seedlings were cultured in the medium which added the different concentrations of TC, and then the contents of chlorophyll and malondialdehyde (MDA), and activity of catalase (CAT), peroxidase (POD) and superoxide dismutase (SOD) of H. cordata leaves were measured. [Results] Compared with the CK, the contents of chlorophyll of H. cordata leaves decreased, and the accumulation of MDA, and the activity of CAT, POD and SOD in H. cordata leaves increased when H. cordata was stressed by different concentration of TC. [Conclusions] The growth, metabolism and physiology of H. cordata were affected by the stress of TC. More importantly, this proved an insight into the effect or harm of undegraded antibiotics in the soil and water in the natural environment to plant growth all the time.

Key words Houttuynia cordata Thunb., Tetracycline, Malondialdehyde, Antioxidant enzyme

1 Introduction

In recent years, the residues of antibiotics, as new organic environmental pollutant, have been threatening the ecological environment and human health, which has become a hot research topic at home and abroad. In European Union, 9 000 t antibiotics were used in 2014, of which 3 000 t were used in Spain, 1 400 t in Italy, 1 400 t in Germany, 779 t in France and 581 t in Poland[1]. The use of the worldwide antibiotics has been the highest in India, China and USA[2]. Because of the broad spectrum and low cost of tetracyclines (TCs), they are widely used in disease prevention, feed addition and promotion of livestock and poultry growth. And the excreted manure is widely used as organic fertilizer for agricultural production. Coupled with the use of TCs to treat human and animal diseases, the application of the TCs is increasing worldwide[1]. China has become one of the countries that produce and consume the TCs in the world[3].

TCs are detected in all samples in Hongze Lake, with the contents of TCs ranging from 1.35 to 25.43 μg/kg[4]. Many studies on evaluating the contamination profiles of TCs in the drinking water sources of the Yangtze River have been described. It has been reported that the maximum concentration of TC is found in dry season as 11.16 ng/L[5]. TCs, which come from medicine, breeding and agriculture, are absorbed by humans and animals, and most of them are excreted in the form of parent compounds, then directly or indirectly spread to the main soil and remain in the soil. TCs disrupt the soil microbial community, and lead to the spread of resistance genes[6]. On the other hand, they will be absorbed by vegetable crops and remain in agricultural products, thereby threatening human health and ecological safety[7].

There is more and more attention to the environmental pollution problem. Some studies have found that the TC pollution in the soil can lead to its accumulation in vegetable crops, and the content ranges from a few μg/kg to hundreds of μg/kg. The edible parts of some vegetable crops have exceeded the trigger value of the ecotoxic effect of antibiotics in the environment specified by the EU (100 μg/kg)[8-9]. Duan’s research results showed that high TC concentration (5-40 mg/L) inhibited the root elongation of pakchoi seeds[10]. Liu found that activity of peroxidase (POD), superoxide dismutase (SOD) and catalase (CAT) significantly increased in ginger under TC stress[11]. Other studies showed that TCs inhibited growth and biomass accumulation, increased reactive oxygen species (ROS) level, SOD activity and MDA content, and reduced pigment content and CAT activity[12].

POD, SOD and CAT, as antioxidant enzymes, can mediate the stress response of plants to stimuli of the external environments to eliminate the damage of ROS to the plant cells. The changes in the activity of POD, SOD and CAT can reflect, to some extent, their own resistance abilities. In this study, different TC concentrations were used as stress factors, the asepticH.cordataseedlings were used as the test materials, and 1/2 MS medium was used as the growth medium. Then the activity of POD, SOD and CAT, and the contents of chlorophyll (a+b) and MDA ofH.cordataleaves in different stress period were detected. The aim is to explicate whether and howH.cordataplants response to TC.

2 Materials and methods

2.1 Experimental materialsH.cordataseeds: the ears ofH.cordatawere collected on the eighth floor at Key Laboratory for Information System of Mountainous Areas and Protection of Ecological Environment, Guizhou Normal University, Guizhou Province. After drying in natural air, the seeds in the ears were shaken off, and after removing the impurities such as flower buds, the seeds were stored in brown, sealed glass bottles for standby.

AsepticH.cordataseedlings: after disinfection, theH.cordataseeds were evenly sprinkled into the starting medium and put into a light incubator. After the seeds germinated, the seedlings were transferred to the strong seedling culture medium and cultured in the light incubator for about two months. The whole operation process was carried out on the ultraclean bench. The formulation of sterilization agent and culture medium was according to the method of Yeetal[13].

2.2 Experimental instruments and reagents

2.2.1Instruments. Constant temperature water bath (Tianjin Sterling Instrument Co., Ltd.); constant temperature drying oven (Tianjin City Stewart Instrument Co., Ltd.); AL104 electronic analytical balance (Mettler Instruments); constant temperature light incubator (Tianjin Tester Instrument Co., Ltd.); UV spectrophotometer; centrifuge (Jiangsu Jintan Zhongda Instrument Factory).

2.2.2Reagents. Tetracycline (Shanghai Macklin Biochemical Co., Ltd.); acetone; NBT (Jitai Biotechnology Co., Ltd.); methionine (Shanghai Sifeng Biotechnology Co., Ltd.).

2.3 Experimental designTo prepare the stress medium, sucrose (20 g/L), agar (7 g/L) and MS medium were mixed, and heated with water until dissolving completely. After adjusting pH to about 6.2, the liquid was sterilized in a high-pressure steam sterilization pot (initial 121 ℃ and then 80 ℃, for 20 min). Subsequently, the liquid was transferred to a sterile operation table, added with TC acetone solution that had been passed through 0.22 μm filter, poured into tissue culture bottle and cooled to solidification. After the stress medium was prepared, the seedlings were inoculated into it on the sterile operating table with 6 parallels for each group. The culture conditions were as follows: 23-25 ℃, 1 500-2 000 Lx, 12 h light/12 h dark. The samples were measured once every 6 d. The concentration gradients of TC were 0 (CK), 0.004 (T1), 0.01 (T2) and 0.016 (T3) g/L.

2.4 Methods

2.4.1Analysis of MDA. MDA content was determined using thiobarbituric acid method[14]with some modifications. 10 mL of the MDA extract from freshH.cordataleaves in 0.05 mol/L phosphate buffer solution (pH=7.8) was mixed with 10 mL 0.5% thiobarbituric acid, kept boiled for 20 min, and then cooled quickly. The absorbance of the mixed solution at 450, 532, and 600 nm was measured. The calculation process is as follows:

MDA (μmol/g)=[6.45×(OD532-OD600)-0.56×OD450]×V/(W×1 000)

whereV(mL) is the volume of extracted liquid, andW(g) is the fresh weight of the extractedH.cordataleaves.

2.4.2Analysis of chlorophyll. The chlorophyll content was determined by the method of Kumarietal.[15]with some modifications. Briefly, appropriate weight ofH.cordataleaves was ultrasonically extracted in acetone for 30 min to obtain chlorophyll extract solution. The absorbance of chlorophyll extract solution was measured with spectrophotometer at wavelengths of 663 and 645 nm. The contents of chlorophyll a, chlorophyll b and total chlorophyll were calculated using the following formulas:

Chlorophyll a (mg/g)=(12.7×OD663-2.69×OD645)×V/(W×1 000)

Chlorophyll b (mg/g)=(22.9×OD645-4.68×OD663)×V/(W×1 000)

Chlorophyll (mg/g)=Chlorophyll a+Chlorophyll b

whereV(mL) represents the volume of the chlorophyll extract solution, andW(g) represents the weight ofH.cordataleaves used to extract the chlorophyll.

2.4.3Analysis of POD activity. POD activity was measured using guaiacol method[16]with some modifications. FreshH.cordataleaves was added with an appropriate volume of phosphate buffer (100 mmol/L, pH=6), ground in ice bath, transferred to centrifuge tube, centrifuged and filtered in success. The operation above was repeated once, and the extract solution of the two times was merged and diluted with phosphate buffer to 25 mL. The reaction mixture solution was prepared by dissolving guaiacol and hydrogen peroxide with phosphate buffer. The absorbance of the solution was measured at 470 nm. A unit of POD activity is expressed as an absorbance change of 0.01 per minute. The calculation process of POD activity is as follows:

POD [U/(g·min)]=ΔOD470×VT/(0.01×t×W×VS)

whereVT(mL) represents the volume of POD extract,VSrepresents the volume of POD for experiment, andW(g) represents the mass of sample used for POD extraction.

2.4.4Analysis of CAT activity. CAT activity was measured by ultraviolet absorption method[17]with some modifications. CAT of freshH.cordataleaves was extracted using 50 mmol/L phosphate buffer (pH=7), and the extract solution was centrifuged, filtered, and diluted with phosphate buffer to 25 mL. The crude CAT was reacted with 200 mmol/L H2O2, and then its UV absorption was measured at 240 nm. The change ofOD240per minute was regarded as a CAT activity unit. The CAT activity is calculated as follows:

CAT [U/(mg·min)]=(ΔOD240×VT)/(0.1×VS×t×W)

whereVT(mL) represents the volume of the CAT extract,VS(mL) represents the volume of the CAT used in the experiment, andW(g) represents the weight of the sample used to extract the CAT.

2.4.5Analysis of SOD activity. The SOD activity was measured referring to Raoetal.[18]with some modifications. Briefly, a certain amount ofH.cordatafresh leaves was weighed, added with an appropriate volume of phosphate buffer (50 mmol/L, pH=7.8), grinded and centrifuged, and the supernatant was collected and diluted to 25 mL as the crude enzyme solution. 0.24 mL crude enzyme solution was mixed with 0.7 mL methionine (130 mmol/L), 0.7 mL nitrogen blue tetrazole (NBT) (750 μmol/L), 0.7 mL EDTA-Na2(100 μmol/L), 0.7 mL riboflavin (20 μmol/L) and 1 mL H2O, and the mixed reaction solution was placed into a light incubator for 20 min at 25 ℃ to inhibit the photoreduction reaction of nitrogen blue tetrazole (NBT) by 50% as an SOD activity unit. The calculation process of SOD activity is as follows:

SOD [U/(g·h)]=[(OD0-OD560)×Vt×60]/(OD0×0.5×W×Vs×15)

whereOD0is the absorbance of light control group,Vt(mL) is the volume of the extracted liquid,Vs(mL) is the volume of the SOD used in the experiment, andW(g) is the fresh weight of the extracted leaves.

2.5 Data statistics and analysisThe experiment was conducted with at least three replicates for each treatment. Means and standard error were calculated for each treatment. Significant differences were determined by using an analysis of variance followed by a Tukey’s Highly Significant Difference Test. Data mapping were performed by Excel 2013 software and all statistical analysis was done with the GraphPad prism7 software and difference withPvalue less than 0.05 was considered to be significant.

3 Results and analysis

3.1 Effect of TC on MDA inH.cordataleavesMDA is an important indicator reflecting the degree of damage to the membrane system and plant resistance. When plants are subjected to a stress environment, a large number of MDA, one of the membrane peroxidation products, is produced[19]. It can be seen from Fig.1 that, compared with CK, the MDA content ofH.cordataleaves increased with increase of TC concentration. On 6 and 12 d, the MDA content of T1 and T2 was slightly greater than CK, respectively, but the MDA content of each treatment was significantly greater than CK on 18, 24 and 30 d, respectively (P<0.05). The MDA contents of T3 was always significantly greater than that of CK (P<0.05), and greater than the other treatments (P<0.05). The MDA content of each treatment increased with the increase of the stress time. On 30 d, the MDA content of theH.cordataleaves reached the maximum in each treatment.

Note: Different lowercase letters above the columns of the same stress time in the figure indicate significant differences at P<0.05 level. The same below.

3.2 Effect of TC on chlorophyll inH.cordataleavesChlorophyll is the main pigment for photosynthesis in plants. Antibiotics of the soil can be absorbed by the plants and cause chemical denaturation of plant chlorophyll[18], resulting in decrease of plant chlorophyll content. As shown in Fig.2 and Fig.3, under different concentrations of TC, the chlorophyll content ofH.cordataleaves in each treatment significantly decreased compared to that of CK. Obviously, with the increase of stress time, the chlorophyll content ofH.cordataleaves in T3 significantly decreased on 18, 24 and 30 d (except on 6 and 18 d) (P<0.05), the chlorophyll content ofH.cordataleaves in T2 significantly decreased compared to that of CK on 6, 18, 24 and 30 d, except on 12 d (P<0.05), and the chlorophyll content ofH.cordataleaves in T1 significantly decreased compared to that of CK on 6, 12, 24 and 30 d, except on 18 d (P<0.05). The chlorophyll content ofH.cordataleaves in different TC treatment group more and more obviously decreased from 24 to 30 d than that of CK.

Note: CK, control check; T1, 0.004 g/L TC; T2, 0.01 g/L TC; T3, 0.016 g/L TC; 6 d, 6 days; 30 d, 30 days.

Fig.3 Effect of TC on chlorophyll content in leaves of Houttuynia cordata Thunb. (n=3)

3.3 Effect of TC stress on POD inH.cordataleavesPOD, a type of oxidase, is widely present in various plant organisms and can oxidize various hydrogen donors, such as phenols, amines, ascorbic acid, indole and inorganic ions[20]. It can be seen from Fig.4 that the POD activity in the leaves ofH.cordatain each stress treatment was greater than that of CK. The POD activity of TC treatments (T1, T2 and T3) was higher than that of CK. The POD activity of T1 was the highest among the treatments and was significantly higher than that of CK (P<0.05).

Fig.4 Effect of TC on POD activity in leaves of Houttuynia cordata Thunb. (n=3)

3.4 Effect of TC on CAT inH.cordataleavesCAT, as an oxidoreductase, is widely present in plants, animals and microorganisms. CAT can promote H2O2decomposition, remove H2O2in the body and prevent peroxidation. It can be seen from Fig.5 that except for the T1 on 12 and 24 d, the CAT activity of each treatment was significantly higher than that of the CK (P<0.05). The CAT activity of CK reached the maximum on 12 d; the CAT activity of treatments T1 and T2 reached the maximum on 24 and 18 d respectively; while the CAT activity of T3 showed an upward trend, and was the maximum on 30 d, and it was always significantly greater than that of the CK (P< 0.05).

Fig.5 Effect of TC on CAT activity in leaves of Houttuynia cordata Thunb. (n=3)

3.5 Effect of TC stress on SOD in leaves ofH.cordataSOD, one of the participants in the ROS scavenging reaction, plays an important role in the ROS scavenging reaction process. SOD is the core of the antioxidant enzyme systems and can remove superoxide anion free radicals in organisms and protect. The protections of the free radicals to the body are widely distributed in the cells of plants, animals and microorganisms. As shown in Fig.6, except for the SOD activity of the T3 on 12 and 18 d, which was lower than that of the CK (P<0.05), the SOD activity of the other treatments were greater than that of the CK. The SOD activity of the T1 and the T3 showed an overall upward trend. The SOD activity of the T1 was significantly greater than that of the CK (P<0.05). The SOD activity of the T3 was significantly greater than that of the CK on 18 d (P<0.05). Except on 12 and 30 d, the SOD activity of the T2 at other time was significantly greater than CK (P<0.05).

Fig.6 Effect of TC on SOD activity in leaves of Houttuynia cordata Thunb. (n=3)

4 Discussion

The ROS produced by cell metabolism in the normal state of organisms does not cause serious harm to organisms, but can cause biological damage under adverse conditions. The scavenging systems of SOD, POD and CAT can effectively inhibit the ROS’s damage of the body[21-22]. Therefore, the defense enzymes (protective enzymes) in the plant will be active under biotic or abiotic stress, and the enzyme activity will increase or decrease. The SOD, POD and CAT can mediate the stress response of the ROS to plants, and the changes in the activity of these enzymes can reflect their own resistance ability to some extent. After the plant is stressed, the ROS content in the body will continue to accumulate. Furthermore, excessive ROS will cause the peroxidation of biofilm lipids to form harmful substances, and destroy the chloroplast structure and weaken the photosynthesis abilities of the plants[23].

To a certain extent, the SOD activity will increase with the intensification of low temperature stress, and if the critical value of SOD is exceeded, its activity will decrease with the intensification of stress. In this study, after exposed to TC for 30 d, the SOD activity of T1 is greater than that of T2 and T3, which is consistent with the results of previous studies. POD activity increased with increasing TC concentration and duration of exposure inVallisnerianatans(Lour.) Hara[28]. Liu found that the antioxidant enzyme system ofTrapabispinosaRoxb. to tetracycline exposure showed higher sensitivity than those ofHydrocharisdubia(Bl.) Backer[29]. In this study, afterH.cordatawas stressed by TC, its own defense response was activated, which significantly increased the SOD, POD, and CAT activity, so as to enhance the adaptability to adversity and maintain metabolic balance in the body. However, the MDA content increased over time. When it comes bigger, it shows that the antioxidant system ofH.cordatacan not effectively alleviate the damage of cell membrane lipid peroxidation, and the ability of antioxidant enzymes to balance peroxidation stress in the body is limited.

Chlorophyll is usually used as an important indicator to assess the toxicity of abiotic stressors[30]. Chlorophyll impairment can reduce the efficiency of photosynthesis and thus affect plant growth and development[31]. Previous studies have shown that TC can decrease the chlorophyll content in yellow lupin seedlings[12, 32]. The study of Qinetal.[33]showed that TC addition significantly inhibited the net photosynthetic rate and chlorophyll content. High oxidative stress can also influence the biosynthesis of chlorophyll[27]. At the same time, the POD-H2O2decomposition system can participate in the degradation of chlorophyll and accelerate leaf aging[34]. Under the stress of various concentrations of TC, the POD activity increased, which was consistent with the decrease of chlorophyll content ofH.cordata. In this study, the chlorophyll content was higher in the CK than that in the other treatments. TC stress can reduced the chlorophyll content of leaves and affect the photosynthesis ofH.cordata. The results are consistent with previous studies.

5 Conclusions

The content of chlorophyll decreased, and the accumulation of MDA and the activity of CAT, POD and SOD increased whenH.cordatawas stressed by different concentrations of TC. This suggested that the growth, metabolism and physiology ofH.cordatawere affected by the stress of TC. More importantly, this proved an insight into the effect or harm of undegraded antibiotics in the soil and water in the natural environment to plant growth all the time.